Mutants of an esterase (PFE) from Pseudomonas fluorescens were
generated using Stratagenes mutator strain, Epicurian Coli
XL1-Red. One variant (A209D / L181V) stereoselectively hydrolyzed a
sterically-hindered 3-hydroxy ester, which was not accepted as substrate by the
wild type. After several mutation cycles, mutants were assayed by plating the
esterase-producing colonies onto minimal-media agar plates containing the
3-hydroxy ethyl or glyceryl ester and indicators.

Lipases, as well as esterases, accept a wide range of non-natural esters and
also exhibit high activity in organic media.1,2 However, for
sterically hindered substrates, these enzymes usually fail.

Figure
1

In previous articles, we were able to resolve aliphatic3 and
arylaliphatic4 3-hydroxy esters using commercial lipases or
esterases with good to excellent optical purities. Still, the esters could
not be resolved for the 3-hydroxy ester 1 bearing two methyl groups at
carbon 4 (Figure
1). None of the 18 lipases and 2 esterases tested showed any activity.
We considered several options: screen for new enzymes (tedious and time
consuming), alter the reaction conditions (e.g. change the solvent system
or acyl donor), or evolve new enzymes by mutagenesis.

The last strategy required the gene encoding the enzyme and an efficient
expression system. Fortunately, our lab had a gene encoding a PFE from Pseudomonas
fluorescens, which was expressed well in E. coli using a
rhamnose-inducible promoter. Since the structure of PFE is not known, positions
for site-directed mutagenesis are difficult to predict. Alternatively, a random
mutagenesis of the gene combined with an assay system also allows detection of
the desired variants.

Recent methods are described for the directed evolution of enzymes by random
mutagenesis using error-prone PCR or DNA-shuffling.5,6 For both of
these methods, ligation of the PCR products is critical. In addition, the
mutation bias must be optimized. We chose to use the mutator strain, Epicurian
Coli XL1-Red (lacking DNA repair mechanisms), which should result in
random mutations generated within a clone of interest.7

To identify the desired variants from the numerous clones produced by random
mutagenesis, we supplemented agar plates containing E. coli colonies with
rhamnose, substrate 1, and indicators. A color change, caused by the release of
substrate 1s corresponding acid, indicated hydrolysis. To further select for
the mutants, the corresponding glycerol ester 2 was introduced. When this ester
is hydrolyzed, the carbon source glycerol is released, thereby speeding up
bacterial growth and producing active esterases on minimal media. With this
strategy, we identified an esterase variant capable of stereoselectively
hydrolysing the sterically-hindered 3-hydroxy ester 1.8 This compound
can serve as a building block in the synthesis of Epothilones, a new class of
macrolides showing taxol-like biological activity.9

Mutation and Screening

Figure
2

In Figure
2, plasmids isolated from E. coli colonies produced during
mutation cycles using XL1-Red competent cells were transferred into a
non-mutator E. coli host, then plated and incubated on LB/Amp agar
plates. From these master plates, colonies were replica plated onto minimal-media
agar containing rhamnose (to induce esterase production), substrates
1 or 2, and indicators (crystal violet and neutral red). Upon hydrolysis
of substrates 1 or 2, the pH in the microenvironment of the colonies decreased
and red spots are formed (from the indicator).

We achieved a good compromise between fast bacterial growth and reliable
detection of positive variants by using plates with minimal media. From
approximately 750 colonies, several putative mutant clones were identified based
on the red color that developed on the agar plates after the colonies were
incubated for 2 to 6 days at 37C.

The plasmids were isolated using colonies from the master plates and then
transformed back into a non-mutator host. After the colonies were cultivated for
3 hours and induced with rhamnose for 4 hours, the esterase produced was
isolated by sonification and subjected to preparative biotransformation. When
the esterases capable of hydrolysing the 3-hydroxy ester 1 were sequenced, one
variant contained two point mutations (A209D and L181V). In the indicator
assay, the clone that produced this variant also gave the strongest red color
and largest colony size.

The esterase gene is 843 bp long, and the double mutant was isolated after
three mutation cycles. Each cycle is approximately 30 generations and the
spontaneous mutation rate should yield approximately 1 mutation per 2,000 base
pairs every 30 generations. This PFE mutant was subjected to another mutation
cycle using XL1-Red competent cells, and approximately 9500
colonies were
investigated. From these, 12 clones were selected, and the esterase was produced
and subjected to biotransformation reactions; however, the stereoselectivity did
not improve.

Biotransformation

The esterases (wild type and selected mutants) were subjected to the
preparative hydrolysis of substrate 1 in phosphate buffer (50 mM, pH 7.5) at 40C.
As expected, no reaction was observed either with the wild type (PFE) or in the
absence of enzyme. In contrast, by using variant A209D/L181V, stereoselective
hydrolysis of substrate 1 occurred, resulting in what is 25% enantiometric
excess (%ee) (determined by gas chromatography using a chiral column) for the
remaining ester. The stereoselectivity was further confirmed by determining the
optical rotation of the substrate and the reaction product. The remaining ester
substrate gave an [a]D20 of + 0.97 , and the produced acid, as
expected, rendered a contrary sense of rotation of [a]D20= - 11.01
after a 10-day reaction time. Mutants derived from the second mutation cycle
exhibited less stereoselectivity compared to the variant, A209D/L181V. The low
stereoselectivity observed in the esterase-catalyzed resolution of substrate 1
relates to the generally observed weak stereoselectivity of PFE.10

Conclusions

The results clearly demonstrate that the XL1-Red mutator strain is efficient
as an alternative method to direct the evolution of enzymes. Besides being easy
to use, XL1-Reds mutation rate compares well to error-prone PCR, while
excluding the problems associated with PCR ligation. Because the assay does not
require special substrates, such as chromophoric esters, a large number of
clones can be quickly identified by their surrounding red color. Additionally,
only a few mutation cycles are necessary to identify an esterase variant, which
hydrolyzes a sterically-hindered substrate. We expect that direct
ed evolution
using the XL1-Red mutator strain will easily transfer to other experiments with
problems related to biotransformation.

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